PHYSICIAN'S MANUAL 
OF 
LABORATORY DIAGNOSIS 
Compiled by 
DR. J. W. LOWELL 
For 
CHICAGO SURGICAL and ELECTRICAL CO. 
CHICAGO PHYSICIAN'S MANUAL 
OF 
LABORATORY DIAGNOSIS 
Compiled by 
DR. J. W. LOWELL 
For 
CHICAGO SURGICAL and ELECTRICAL CO. 
CHICAGO INTRODUCTION. 
It is the purpose of this manual to give, in as simple and as 
brief a manner as possible, those more common laboratory tests, 
which the physician himself can and should make. For alterna- 
tive methods, for a discussion of the interpretation of laboratory 
findings and for methods not here given, the reader is referred 
to larger books on laboratory diagnosis, a few of which are 
listed herewith : 
Webster's Diagnostic Methods, P. Blakiston's Son & Co. 
Emmerson's Clinical Diagnosis, J. P. Lippincott Co. 
Wood's Chemical and Microscopical Diagnosis, D. Appleton 
& Co. 
Hawk's Practical Physiological Chemistry, P. Blakiston's 
Son & Co. 
For assistance in the revision of this manual we are indebted 
to Dr. Oscar T. Schultz, director of the Nelson Morris Institute 
for Medical Research of the Michael Reese Hospital, Chicago. 
Chicago Surgical and Electrical Co. CONTENTS. 
Page 
I. Urine: 
Apparatus 6 
Reagents 7 
Qualitative Examination: 8 
Color " 
Transparency " 
Reaction 9 
Specific Gravity .' '1 
Total Solids " 
Albumin " 
Sugar 10 
Blood " 
Pus " 
Bile " 
Acetone 11 
Diacetic Acid " 
Indican " 
Microscopic Examination: 12 
Casts " 
Crystals 13 
Cells " 
Bacteria " 
Quantitative Examination: . . . . 14 
Acidity " 
Ammonia " 
Urea 15 
Chlorides " 
Sulphates 16 
Phosphates " 
Albumin 11 
Sugar " 
Clinical Importance 17 
II. Examination for Micro-organisms: 19 
Apparatus " 
Reagents 20 
Sterilization " 
Culture Media: 21 
Bouillon " 
Agar 22 
Gelatine 11 
Loeffler's Serum Media " 
Inoculation " 
Incubation 23 
Preparation of Smear " 
Types of Bacteria " 
Gram Negative Bacteria 24 
Gram Positive Bacteria " 
Micrococcus Intracellularis 25 
Micrococcus Catarrhalis 11 
Micrococcus Gonorrheae 11 
3 Page 
Bacillus Coli 26 
Bacillus Typhosus " 
Bacillus Dysenteriae 11 
Bacillus Pyocyaneus " 
Bacillus Influenzae 27 
Bacillus Fusiformis " 
Staphylococcus " 
Streptococcus " 
Pneumococcus 28 
Micrococcus Tetragenus " 
Bacillus Tetani " 
Bacillus Tuberculosis 29 
Bacillus Diphtheriae " 
Bacillus Anthracis 30 
Entamoeba Buccalis " 
Entamoeba Histolytica 32 
Widal Test " 
India Ink Method for Treponema Pallidum 33 
III. Sputum: 35 
Color " 
Consistency 
Odor 36 
Special Characteristics " 
Outline for Routine Examination 37 
Bacteria Commonly Found in Sputum " 
IV. Blood: 38 
Apparatus " 
Reagents " 
Normal Blood: " 
Erythrocytes 11 
Lymphocytes " 
Leucocytes " 
Transitional Leucocytes 39 
Pathological Cells Found in Blood: " 
Microcytes " 
Macrocytes " 
Normoblasts " 
Megaloblasts 11 
Poikilocytes " 
Myelocytes " 
Hemoglobin Content " 
Enumeration of Red Corpuscles 40 
Enumeration of White Cells 41 
Color Index 42 
Microscopic Examination of Stained Smear 43 
Differential Diagnosis of Blood Diseases 44 
V. Spinal Fluid: 46 
Apparatus " 
Reagents " 
Cell Count " 
Noguchi Test " 
4 Page 
Koss-Jones Test 46 
Bacteriological Examination 47 
VII. Feces: 48 
Apparatus " 
Reagents 11 
Qualitative Examination: 11 
Lactic Acid 11 
Blood 11 
Boas-Oppler Bacillus li 
Quantitative Examination: " 
Free Hydrochloric Acid " 
Total Acidity " 
Combined Hydrochloric Acid 49 
VI. Stomach Contents: 50 
Apparatus " 
Reagents 51 
Blood " 
Ova " 
Parasites " 
5 I.-URINE 
Apparatus Required for Uranalysis. 
Test tubes, 6 inch. 
Test tube brush and rack. 
Urinometer. 
Doremus' ureometer. 
Einhorn saccharometer. 
Burette, 25 cc., graduated in 0.1 cc. 
Glass funnel, 4 inch. 
Graduate, 100 cc. 
Nest of beakers, assorted. 
Glass tubing of different sizes (small amount). 
Bunsen burner or alcohol lamp. 
Electric test tube boiler. 
Electric centrifuge with plain and graduated tubes. 
Slides and cover slips. 
Microscope with accessories. 
Reagents Required for Uranalysis. 
Concentrated nitric acid-sodium nitroprusside (^ ounce). 
Concentrated hydrochloric acid-ferric chloride, 10% solution. 
Concentrated ammonia. 
Glacial acetic acid. 
Peroxide of hydrogen. 
Cloroform. 
Gum guaiac. 
Potassium hydroxide (dry). 
Formalin. 
Filter paper. 
Red and blue litmus paper. 
1% Phenolphthalein solution in 95% alcohol. 
N/10 Sodium hydroxide (this should be purchased already pre- 
pared). 
Roberts' Reagent: 
(For qualitative albumin test) 
Nitric acid, concentrated 1 part 
Magnesium sulphate, saturated solution 5 parts 
Silver Nitrate Solution: 
(For quantitative chloride determination) 
Silver nitrate 5 grams 
Distilled water 100 c c. 
Keep in a dark bottle 
Rice's Solution: 
(For quantitative determination of urea) 
Solution No. 1. Sodium hydroxide 100 grams 
Distilled water qsad 250 c c. 
Solution No. 2. Bromine (pure) 10 c c. 
Potassium bromide 30 grams 
Distilled water qsad 250 c c. 
6 Haines' Solution: 
(For sugar test) 
Cupric sulphate 12 grams 
Potassium hydroxide 45 grams 
Glycerine 90 c c. 
Distilled water qsad 1000 c c. 
Esbach's Reagent: 
(For quantitative albumin test) 
Picric acid 10 grams 
Citric acid 20 grams 
Distilled water 1000 c c. 
Barium Chloride Solution: 
(For quantitative determination of sulphate) 
Barium chloride 20 grams 
Hydrochloric acid, concentrated 5 c c. 
Distilled water 80 c c. 
Uranium Nitrate Solution: 
(For qantitative determination of phosphates) 
Uranium nitrate 5 grams 
Distilled water 100 c c. 
7 QUALITATIVE EXAMINATION. 
Qualitative Examination. 
The routine qualitative examination of the urine should in- 
clude the determination of color, transparency, reaction, specific 
gravity, presence or absence of albumin or sugar and the micro- 
scopical examination. To these are to be added, in special cases, 
the determination of bile, blood, acetone bodies, indican, etc. 
Color. The normal color is pale straw or amber. The depth 
of color is increased in all conditions which cause a decreased 
output. Abnormal colors may be due to the presence of abnormal 
constituents. Halliburton has tabulated the chief variations in 
color as follows: 
Color. 
Nearly colorless. 
Dark yellow to 
brown-red. 
Milky. 
Orange. 
Red or reddish. 
Brown to brown- 
black. 
Greenish . yellow, 
greenish-brown, 
approaching 
black. 
Dirty green or 
blue. 
Brown - yellow to 
red - brown be- 
coming blood- 
red upon adding 
alkalie. 
Cause of Coloration. 
Dilution, or diminution of 
normal pigments. 
Increase of normal, or oc- 
currence of pathological, 
pigments. Concentrated 
urine. 
Fat globules. 
Pus corpuscles. 
Excreted drugs. 
Hematoporphyrin. 
Unchanged hemoglobin. 
Pigments in food (logwood, 
madder, bilberries, fuch- 
sin). 
Hematin. 
Mathemoglobin. 
Melanin. 
Hydrochinol and catechol. 
Bile-pigments. 
A dark blue scum on sur- 
face, with a blue deposit, 
due to an excess of in- 
digo-forming substances. 
Substances contained i n 
senna, rhubarb and che- 
lidonium which are in- 
troduced into the system. 
Pathological Condition. 
Nervous conditions; hy- 
druria, diabetes insipidus, 
granular kidney. 
Acute febrile diseases. 
Chyluria. 
Purulent diseases of the 
urinary tract. 
Santonin, crysophanic acid. 
Hemorrhages, or hemoglo- 
binuria. 
Small hemorrhages. 
Methemoglobinuria. 
Melanotic sarcoma. 
Carbolic-acid poisoning. 
Jaundice. 
Cholera, typhus; seen es- 
pecially when the urine 
is putrefying. 
Transparency. Freshly excreted normal urine is clear. A 
urine originally clear may become cloudy on standing through 
the development of bacteria, or the deposition of phosphates or 
urates. Turbidity of the freshly voided urine may be due to 
excessive amounts of phosphates or urates or to bacteria, pus or 
blood. The cause of the cloudiness will be established by the 
microscopic examination. 
8 Reaction. The usual normal reaction is acid. It may be 
neutral or alkaline. To determine the reaction, dip a strip of 
litmus paper in the urine. Blue litmus will be changed to red 
by an acid urine, red litmus to blue by an alkaline urine and 
neither will be changed by a neutral urine. 
Specific gravity. The normal specific gravity of urine is 
1.012 to 1.025, varying according to the amount of urine secreted 
in a day. How to find specific gravity: The specific gravity is 
taken by means of a urinometer. The jar (as shown in the cut) 
Urinometer 
is filled with urine and the urinometer is placed within it. The 
reading is made by holding the jar at the level of the eyes and 
reading the number on the graduated index of the instrument 
at the level of the urine in the jar. The urinometer should float 
freely in the urine. 
Total solids. The solid content of the urine is estimated from 
the specific gravity by the formulae given. The normal total solid 
for 24 hours in about 60 grams. 
Trapp's formula (parts of solids per litre of urine) : 
Multiply the last two figures of the specific gravity 
by 2. Example : Specific gravity equals 1023. 23x2 
equals 46 parts of solids per litre of urine. 
Metz's formula (total solids per 24 hours) : 
Multiply the last two figures of the specific gravity 
by 0.00233 and multiply this product by the total 24 
hour urine quantity in cubic centimeters. Example: 
Specific gravity equals 1024. Total urine equals 
1500 cc. 24x0.002333x1500 equals 87.27 grams of 
solids in the total 24-hour excretion. 
Albumin. Heat and acetic acid. If the urine is cloudy, it 
should be filtered or centrifuged. Fill a test tube about two- 
thirds full of urine and add five drops of 2% acetic acid. Heat 
the urine at the top of the column by holding the tube at the 
9 bottom and directing the flame against the upper part of the 
tube. After boiling, add two or three more drops of the acid 
and examine against a black or dark background, for the presence 
of a cloud in that portion of the urine which has been heated. 
Ring test. Place 5 cc. of Roberts' reagent in a test tube and 
stratify over it a small quantity of urine by allowing the urine to 
flow slowly down the side of the inclined tube from a pipette. A 
white ring at the zone of contact indicates the presence of 
albumin. 
Electric Test Tube Boiler 
Sugar. Haines' test. Place 5 cc. of Haines' solution in a test 
tube and heat to boiling. Add six to eight drops of the urine 
and again heat to boiling. A yellow or brownish red precipitate 
is formed if sugar is present. 
Blood. Guaiac test. To exclude a reaction due to pus, the 
urine should be boiled for a few seconds and then cooled. Make 
a freshly prepared alcoholic solution of guaiac (approximately 1 
part of the latter to 60 parts of 95% alcohol). Place 5 cc. of the 
boiled and cooled urine in a test tube and acidify with acetic acid. 
With a capillary pipette, add the guaiac solution until turbidity 
results. Then add hydrogen peroxide drop by drop. A blue 
color indicates the presence of blood. 
Pus. Potassium hydroxide test. Centrifugalize the urine 
and pour off the fluid from the sediment. To the latter, add a 
small piece of solid potassium hydroxide and stir with a glass 
rod. If pus is present, the sediment becomes slimy, gelatinous 
and tough. Fresh pus gives the blue color of the guaiac test 
before boiling, but not after boiling. 
Bile. Filter a small amount of urine through a small filter 
paper. Allow a drop of concentrated nitric acid to fall on the 
moist filter paper. A green ring forms about the acid drop if 
bile is present. 
10 Acetone. Make a weak solution of sodium nitroprusside by 
dissolving a small crystal in 5 cc. of water. Add 1 to 2 cc. of 
this solution to 5 cc. of the urine and render the mixture alkaline 
with potassium hydroxide solution. A ruby red color results. 
Add enough glacial acetic acid to bring about an acid reaction. 
The red color is intensified if acetone is present and disappears 
if acetone is absent. 
Diacetic acid. To 5 cc. of urine in a test tube, add 10% 
ferric chloride drop by drop. If a heavy precipitate forms, filter 
and add a few more drops of the ferric chloride solution to the 
filtrate. A Burgundy red color develops in the presence of 
diacetic acid. If the patient has been taking phenol, salicylates, 
aspirin, acetanilide or antipyrin, the urine gives a dark red but 
more purple color with ferric chloride. 
Indican. To about 5 cc. of urine in a test tube, add an equal 
amount of concentrated hydrochloric acid. Now add 2 to 3 drops 
of peroxide of hydrogen and 3 to 4 cc. of chloroform. Shake 
well and allow to stand. In normal urine there may be a very 
pale blue in the chloroform indicating a trace of indican. This 
blue color when well marked should be considered pathologic. 
11 Microscopic Examination of the Urine. 
The following rules should be observed: 
1. Never use old urine that has undergone decomposition. 
2. Be sure the specimen was collected in a clean receptacle 
and that it is not contaminated with dust, etc. 
3. Make a careful examination with the low power objective 
before using the high power. 
4. Keep the iris diaphragm nearly closed to cut down the 
light. 
Electric Centrifuge 
Method for Making a Preparation for Microscopic Examination. 
Fill a 15 cc. centrifuge tube with urine and rotate it five 
minutes 1800 revolutions per minute. Pour off the clear urine 
(which may be used for the albumin test) and save the precipitate, 
(dean a slide and cover glass and place a drop of the sediment 
on the slide. Now place a cover on it and examine in the usual 
way. The microscopical examination pays especial attention to 
crystals, casts, cells and bacteria. 
Casts. 
Casts are moulds of the kidney tubules. They are usually 
associated with the presence of albumin, but either may occur 
separately. Casts may be hyalin, granular, epithelial, waxy, fatty 
or they may be composed of pus cells, blood, or bacteria. A cast 
should be fairly uniform in diameter and the ends should be 
rounded or abrupt, no tapering. 
Hyalin casts are almost transparent and have the appear- 
ance of ground glass if the light is properly adjusted. 
Granular casts are more opaque than hyalin casts and contain 
granules, which are present throughout the entire substance of 
the casts. 
Epithelial casts are made up of renal epithelial cells, which 
are grouped together to form a short cylinder, the cells being 
held together by a hyalin or mucoid matrix. 
Waxy casts are rare. They are denser and more refractile 
than hyaline casts. 
12 Fatty casts are hyalin, granular, or epithelial casts, which 
contain refractile globules of fat. 
Blood, pus or bacterial casts have the composition indicated 
by the name. In the blood casts, the red corpuscles usually show 
various stages of disintegration. In the pus casts, the cells are 
smaller than in the epithelial variety. Bacterial casts are like 
granular casts but are more closely, finely, and uniformly 
granular. 
Crystals. 
The characteristics of the crystals usually seen in the urine 
are as follows: 
Urates appear as brick dust deposit. 
Uric acid is commonly seen as rhomboidal plates, having a 
tendency to cluster. The color is usually yellow, reddish brown 
or brown. 
Calcium oxalate appears as small envelope shaped crystals. 
Triple phosphate appears as coffin lid crystals. 
Ammonium urates have the appearance of thorn apples of 
yellowish or brown color. 
Cells. 
Epithelial cells. These are always present in moderate num- 
bers in the urine. At times, under inflammatory conditions, their 
number is greatly increased. They are of no great clinical im- 
portance and are outlined purely for differential purposes. 
Squamous large flat cells. They are from the bladder, 
vagina and urethra. 
Irregular cells, caudate and columnar, are from the deep 
layers of the urinary tract. 
Round cells from the deep layers of the pelvis of the 
kidney or bladder. 
Blood cells. Blood cells in the urine are of great significance 
in some disorders. They appear as biconcave disks of a reddish 
yellow color. 
Pus cells. Pus cells are polynuclear leucocytes either well 
preserved and normal in appearance, or showing various degrees 
of degeneration and disintegration. In the unstained preparation, 
they are larger than red corpuscles, are colorless, rather hyalin 
and granular, and with careful focussing and illumination, the 
lobulated nucleus can be recognized. 
Lymphocytes. In inflammatory processes of longer duration, 
lymphocytes may be present in variable number in the urine. 
They are of about the size of a red corpuscle, have a ground glass, 
finely granular appearance, and the individual cell is made up, 
largely, of the single round nucleus. 
Bacteria. 
The following are those most commonly found in the urine: 
Gram positive bacteria: staphylococci and streptococci. 
Gram negative bacteria: gonococcus, colon bacillus and 
typhoid bacillus. 
Acid fast bacteria: tubercle bacillus, smegma bacillus. 
13 QUANTITATIVE EXAMINATION. 
Quantitative Examination. 
Only some of the more simple determinations, readily per- 
formed by the physician with the apparatus listed, are given. 
The methods given do not pretend to exact scientific accuracy, 
but have the advantage of simplicity and give results of sufficient 
accuracy for clinical work. 
Burette 
Acidity. 
Fill the burette with N/10 sodium hydroxide solution. Note 
the quantity of fluid in the burette, always reading the lowest 
point of the meniscus. Place 10 cc. of urine in a beaker and add 
2 to 3 drops of 1% alcoholic solution to phenolphthalein. Now 
add N/10 sodium hydroxide solution from the burette slowly to 
the urine, stirring constantly until a light pink color persists. 
The urine is now neutral. Now note the amount of sodium 
hydroxide used in neutralizing 10 cc. of urine and multiply the 
number of cc. used by 10. The result will represent the degree 
of acidity. The normal acidity expressed in this manner varies 
from 15 to 30. 
Ammonia. 
Neutralize 10 cc. of urine in the manner as explained under 
determination of acidity. The urine used for determining the 
acidity may be used for the determination of ammonia. Now 
neutralize, separately, 10 cc. of 20% formalin with N/10 sodium 
hydroxide, using phenolphthalein as an indicator (as above). Mix 
the two solutions, note carefully the burette reading, and titrate 
14 the solution to permanent pink color. Again note the burette 
reading to find the quantity of N/10 sodium hydroxide used. 
Multiply this quantity in cc.'s by .0017, which will give the 
number of grams of ammonia in 10 cc. of urine. The normal 
amount is 0.7 grams in a 24-hour day. 
Urea. 
The quantity of urea found in normal urine varies from 20 
to 40 grams in 24 hours. About 80% of the nitrogen excreted is 
in the form of urea. For the quantitative determination of urea, 
fill the Doremus ureometer with equal parts of Rices' solution 
No. 1 and No. 2 (about 10 cc. of each) and 20 cc. of water, taking 
Einhorn's Saccharometer 
Doremus Ureometer 
care to remove any air in the arm. Now fill the pipette with urine 
to the 1 cc. mark, pass the end into the ascending arm of the 
instrument, and gradually empty the pipette. Carefully with- 
draw the empty pipette. It will be noted that gas accumulates 
in the upper part of the arm. The quantity of urea may be read 
directly, from the graduations. The tube is graduated to read 
in grams per cc. The reading obtained, multiplied by the total 
24-hour urine quantity in cc., is the total urea output for 24 hours 
in grams. The observed reading multiplied by 100 gives the 
urine percentage. 
Chlorides. 
The amount of chlorides in normal urine should be from 10 
to 15 grams per 24 hours. To determine the amount of chloride 
by means of the centrifuge, put 10 cc. of filtered and albumin-free 
urine into the graduated centrifuge tube, acidify with 1 cc. nitric 
acid and add 4 cc. of a 5% solution of silver nitrate. Invert the 
tube several times and permit to stand 2 to 3 minutes. Now 
place in the centrifuge and rotate 3 minutes at about 1800 revolu- 
tions per minute. Read the amount of sediment. 1 cc. of sedi- 
ment represents .13 grams of sodium chloride and 0.08 grams 
of chlorin in the 10 cc. quantity of urine used. 
15 Quantity of sediment x 0.3 x total 24 hour urine in cc. 
10 
equals grams of sodium chloride excreted in 24 hours. 
Sulphates. 
To 10 cc. of filtered urine in a graduated centrifuge tube, add 
5 cc. barium chloride mixture. Mix well and permit to stand a 
few minutes, then centrifuge three minutes at 1800 revolutions 
per minute. 0.1 cc. of sediment is equal to 0.25% of sulphates 
calculated as sulphuric acid in 10 cc. of urine. 
Quantity of precipitate x 0.25 x total 24 hour urine in cc. 
10 
equals grams of total sulphates as sulphuric acid excreted in 24 
hours. 
The total normal sulphate excretion varies from 1.5 to 3.00 
grams of sulphuric acid with an average of 2.0 grams. 
Phosphates. 
Place 10 cc. of filtered urine in a graduated centrifuge tube, 
and add 2 cc. of 50% acetic acid and 3 cc. of 5% uranium nitrate 
solution. Mix by inverting the tube several times. Permit it to 
stand a few minutes, then centrifuge for 3 minutes. Each 0.1 cc. 
of precipitate represente 0.0225% of P2 O5 by weight. The 
total 24 hours excretion of phosphates as grams of P2 O5 is 
calculated as follows: 
Quantity of precipitate x 0.0225 x total 24 hour urine in cc. 
~ nF 
equals grams of phosphates as P2 O5 excreted in 24 hours. 
The normal excretion in 24 hours varies from 1 to 5 grams 
of P2 O5. 
Albumin. 
Place 10 cc. of centrifuged urine in a clean graduated centri- 
fuge tube and add 5 cc. of Esbach's solution. Mix the solution 
and urine by inverting several times. Permit them to stand 
several minutes, after which centrifuge for 3 minutes at 1800 
revolutions per minute. Read the percentage of the precipitate. 
Each graduation represents 0.02 grams of albumin. 
Sugar. 
Where there is a strong reaction to the Haines' solution, a 
quantitative sugar test is often advisable, to find the exact amount 
of sugar in the urine. This is accomplished by means of the 
Einhorn saccharometer. Mix 10 cc. of centrifuged urine with a 
piece of fresh yeast the size of a pea. Transfer to the saccharo- 
meter. Be sure all the bubbles are removed from the arm of the 
instrument; this is accomplished by tilting. In the other tube, 
place 10 cc. of normal urine, to which a similar sized piece of 
yeast has been added, to control and exclude the formation of 
gas from the yeast itself. The saccharometer is now placed in the 
incubator at 98 degrees Fahrenheit (37^ degrees Centigrade). 
After 12 hours, the amount of sugar may be read directly from 
the arm of the instrument, as percentage of sugar present. From 
the total 24 hour urine quantity, the quantity of sugar excreted 
can be calculated. 
16 Clinical Importance. 
The clinical importance of some of the more important find- 
ings may be briefly summarized: 
ACIDITY 
Increased 
In meat diet 
Acidosis, etc. 
Decreased 
In fruit and vegetable diet 
Alkaline cystitis 
Administration of certain alkaline drugs. 
AMMONIA 
In the system ammonia neutralizes excessive acidity. 
Importance 
Its relation to acidity is a great guide to the system's 
ability to compensate for increasing acidity espe- 
cially in diabetes millitis. 
UREA 
Increased 
In meat diet 
Tissue destruction as in rapid wasting fevers 
Excessive exercise. 
Decreased 
In a low nitrogenous diet 
destructive diseases of the liver 
Renal insufficiency as in acute nephritis. 
CHLORIDES 
Increased 
Diabetes insipidus 
Dropsy after diuresis 
A rapid increase is noticed with improvement in 
acute fevers. This is often present prior to drop of 
fever. 
Decreased 
Impaired digestive functions 
Acute infectious diseases up to the time of improve- 
ment 
In pneumonia when we have a complete disappear- 
ance of chlorides, the prognosis is serious. 
SULPHATES 
Increased 
In acute fevers 
Meningitis 
Encephalitis 
Rheumatism. 
17 PHOSPHATES 
Increased 
In convalescence from fevers. 
Pulmonary tuberculosis 
Decreased 
In fevers (Zulzer) 
Constant in nephritis (Flesher). 
INDICAN 
Large amounts of Indican indicate 
Intestinal putrefaction as in intestinal indigestion, 
diarrhea, intestinal tuberculosis or lack of hydro- 
chloric acid in the stomach as in carcimona, chronic 
gastritis, etc., or in conditions where there is a lack 
of peristalsis in the intestine 
Putrefaction processes elsewhere in the body 
Gangrene of the lungs as in 
Empyeme 
Advanced pulmonary tuberculosis 
ALBUMEN 
With a definite renal lesion 
All forms of nephritis 
In chronic passive congestions 
Acute congestion of the kidney 
Without definite renal lesion 
Physiological in fever 
Severe exercise 
Heamotogenous as in 
Severe anaemia 
Cachexia 
Syphilis 
Tuberculoses 
Sugar is found in 
Diabetes Millitis 
Destructive lesions of the liver 
Destructive lesions of the pancreas 
Ex-ophthalmic goiter 
Pregnancy 
Acute diseases. 
18 II.-EXAMINATION FOR MICRO-ORGANISMS. 
Apparatus. 
THE MICROSCOPE-A clinical instrument made by one of 
the reliable manufacturers will give satisfaction in this work. 
It should have three objectives, a 16 mm. (2/3 inch), 4 mm. (1/6 
inch), and 2 mm. (1/12 inch), and should be fitted with a con- 
denser and iris diaphragm. 
Electric Incubator 
THE INCUBATOR-An electrically heated incubator should 
be selected. They are much more satisfactory and reliable than 
the old gas heated ones. Choose one which combines safety, 
accuracy and reliability. 
THE CENTRIFUGE-An electric centrifuge is a valuable 
addition, although not absolutely necessary for this work. 
THE WARM STAGE-An electrically heated warm stage 
will be found most convenient. 
PLATINUM LOOP-This can be made by heating the end of 
a glass rod and then inserting the end of a number 22 platinum 
or nichrome wire into it. When cool, the wire, which should be 
about 2 inches long, may be straightened, and then have the end 
bent around a pencil point to form a small loop. 
Three dozen slides. 
One-half ounce of cover slips, No. 1 or No. 2. 
Bunsen burner or alcohol lamp. 
Cover glass forceps. 
Slide box in which permanent preparations may be kept. 
A test tube rack. 
Wire basket for culture tubes. 
19 Stains and Reagents. 
30 co. Carbol fuchsin. 
30 cc. Methylene blue (Loeffler's). 
30 cc. Carbol Gentian Violet. 
30 cc. Saturated aqueous Saffranin. 
30 cc. Xylol. 
30 cc. Lugol's solution. 
20 cc. Canada Balsam. This should be in solution in xylol. 
These may be purchased from some reliable supply house. 
Sterilization. 
Sterilization is a proccess by which all micro-organisms are 
killed. 
This may be accomplished in two ways first by heat, second 
by chemicals. 
Electric Test Tube Boiler 
Sterilizer to be used in con- 
junction with Test Tube Boiler 
Sterilization by Heat, 
Instruments are sterilized by boiling them in water for from 
10 to 20 minutes. 
Glassware is usually sterilized by dry heat. 
This is accomplished by placing it in a so-called sterilizing 
oven and heating it to 150-180 degrees C. for from 30 minutes 
to an hour. 
Culture media are usually sterilized in an auto-clave or an 
Arnold Steam sterilizer. 
An autoclave is composed of a chamber in which the material 
is placed and then subjected to superheated steam at 15 pounds 
pressure for 20 minutes. An Arnold steam sterilizer is composed 
of a chamber in which steam at 100 degrees C. is circulated. 
This forms a very efficient although rather slow method of 
sterilization. 
The open flame is also used to sterilize platinum needles, etc. 
20 GERMICIDES-There are a large number of chemicals which 
will destroy micro-organisms on contact. Some of the most im- 
portant of these are: Solutions of phenol, lysol, and bichloride 
of mercury. These are called germicides. 
ANTISEPTICS-There is another class of chemical solu- 
tions, which inhibit the growth of bacteria and prevent their 
multiplication, although the organisms may not be destroyed. 
These are called antiseptics. 
Laboratory Prophylaxis. 
1. Always think what you do and why. In working with 
organisms heat a platinum wire to red heat to destroy the micro- 
organisms, which may be on it. Permit it to cool so it will not 
destroy the bacteria, which are handled with it. 
2. Always wear a gown when handling infective material. 
Use it for no other purpose. 
3. Always disinfect the hands with 2% lysol. or 5% carbolic 
acid, after working with organisms, and before wiping them on a 
towel. 
4. When you finish with a culture, or with any infective 
material, always kill it with a powerful germicide, or by boiling 
it before throwing it out. In case you spill any infective material 
on the table or floor cover it with 5% lysol solution. Let it stand 
several minutes and then wipe it up. 
It is safest, when working with a highly infectious specimen, 
to handle as few things as possible, before thoroughly washing 
the hands with some good germicide and then with soap and 
water. 
Culture Media. 
If the physician has an Arnold sterilizer or an autoclave at 
hand, he can prepare his own media. This, however, is not prac- 
tical unless considerable quantities are used. Most physicians 
prefer to purchase the media ready for use from one of the 
pharmaceutical houses. The following selection is recommended: 
4 tubes of Loeffler's blood serum media for culturing 
diphtheria. 
4 tubes of glucose agar. 
4 tubes of nutrient gelatin. 
4 tubes of nutrient agar. 
This should be kept in a glass jar. In this way they may be 
kept for several months before becoming too dry for use. 
Nutrient Bouillon. 
Beef extract 10 grams 
Peptone (dry) 10 grams 
Sodium chloride (common salt) 5 grams 
Water 1000 c c. 
Boil until the peptone is entirely dissolved. Test the reaction 
with litmus paper. It will be acid. Now add enough sodium 
hydrate solution (the regular solution used for titrating may be 
used), to render it slightly alkaline to litmus. Boil the bouillon 
again, and filter it until clear. Distribute it in test tubes, 10 cc. 
21 to a tube. Cork with non-absorbent cotton and sterilize in either 
an ordinary steam sterilizer or an autoclave. If the steam steril- 
izer is used, the sterilization must be repeated on three successive 
days. Test in the incubator at 37 degrees C. to assure sterility. 
Nutrient Agar. 
Nutrient bouillon 1000 c c. 
Agar-agar (should be soaked overnight in water). . . 20 grams 
Heat these until the agar is entirely dissolved. Test the 
reaction again. As a rule it is not changed, but remains slightly 
alkaline. Cool the mixture to 60 degrees C. Stir in an egg, which 
has been mixed with a small quantity of water. Filter. To filter, 
tie two thicknesses of cotton flannel over the mouths of six wide- 
mouth 6-ounce bottles. Invert these in a gallon pail, pour in the 
agar mixture, place in the steam sterilizer for 1 hour. Remove 
and allow to cool. The filtered agar will be in the bottles. Dis- 
tribute the filtered agar in tubes, about 10 cc. to a tube, cork with 
non-absorbent cotton and sterilize in the autoclave or steam 
sterilizer. Test in the incubator at 37 degrees to assure sterility. 
Glucose Agar. 
This is prepared from ordinary nutrient agar by adding 1% 
of glucose. 
Nutrient Gelatine. 
Nutrient bouillon 1000 c c. 
Gold Leaf gelatine 100 grams 
Warm to dissolve the gelatine. Neutralize with sodium hy- 
droxide. Clarify and filter as described under nutrient agar. 
Tube and sterilize in the usual way. Test to assure sterility. 
During cultivation gelatine must be kept at room temperature. 
Loeffler's Serum Medium. 
Ox or sheep's blood serum 300 c c. 
Nutrient bouillon 100 c c. 
Glucose 4 grams 
Tube and sterilize in the Arnold steam sterilizer in an in- 
clined position for one-half hour, two successive days. The serum 
will coagulate. Test in the incubator at 37 degrees to assure 
sterility. 
Inoculating a Culture. 
1. Take the platinum loop in you right hand, holding it as 
you would a pencil. Hold it in the flame of the burner until it 
is red hot, and then heat a short distance on the handle. This is 
done to sterilize the wire and also the handle. 
2. Take the culture tube in the left hand and hold it in 
as nearly a horizontal position as possible. 
3. Take a little of the material on the wire loop. 
4. Remove the cotton plug, holding it between the little 
finger and the palm of the right hand. 
5. Flame the mouth of the tube and spread the material 
on the platinum loop over the surface of the medium. 
6. Again flame the mouth of the tube and replace the cotton 
plug. 
7. Sterilize the platinum loop as in No. 1. 
22 Incubating Culture. 
After a culture is made it is placed in a wire basket on the 
shelf of the incubator. The incubator should run at 37 degrees. 
In 12 to 24 hours it will be noted that the path of the loop is 
dotted with small colonies of organisms. If too much material is 
used in making the culture the colonies will coalesce, making it 
impossible to isolate the organisms. It will be noted that most 
have some special cultural characteristics. 
Making and Examining a Stained Smear. 
1. Clean a slide and cover slip. 
2. Flame the platinum wire and permit it to cool. 
3. Take a small amount of the suspected material on the 
platinum loop. 
4. Mix this with a small drop of water which has been 
previously placed on the cover slip which is held in the cover 
glass forceps. 
5. Sterilize the platinum wire in the flame. 
6. Dry the smear high over the flame. 
7. When thoroughly dry pass the cover slip down through 
the flame 2 to 4 times, depending on its size. This is done to 
coagulate the albumin and fix the organisms to the glass. Care 
should be taken not to burn the specimen. A brown color 
indicates burning. 
8. After the preparation is cool, cover it with a few drops 
of the desired stain. Permit it to remain 2 or 3 minutes. 
9. Wash the stain off in water. 
10. Dry between sheets of filter paper and then in the air. 
11. Put a small droplet of Canada balsam on the slide and 
press down the cover slip. Examine with high power and then 
under the oil immersion lense. The smear may be made on the 
slide and examined directly without a cover glass after staining 
and drying. 
Types of Micro-organisms. 
All organisms are divided into three great classes according 
to their shape: 
1. Cocci; ball shaped organisms: staphylococci, clumps of 
cocci; streptococci, chains of cocci. 
2. Baccilli; rod shaped organisms. 
3.- Spirilla; curved or spiral rods. 
These forms are again subdivided according to their staining 
reaction into gram negative and gram positive organisms. 
Gram Stain. 
This is the first special stain used in the differentiation of 
organisms. 
To make a gram stain: After fixing, flood with carbol 
gentian violet, allow to stand for 1 minute, pour off, flood with 
Lugol's solution for 1 minute. Pour off and wash with 95% 
alcohol until all the blue disappears. Wash in water, and counter- 
stain with aqueous suffrunin solution. Wash in water, dry and 
mount. 
23 All gram positive organisms will be purple or deep blue; 
all gram negative organisms will take the counterstain. 
Gram Negative Organisms. 
Gram negative cocci: M. intracellularis, M. catarrhalis, M. 
gonorrheae. 
Gram negative baccilli: B. coli, B. typhosus, B. dysenteriae, 
B. mucosus capsulatus, most frequently from the intestinal tract, 
B. pyocyanous, B. influenzae, B. fusiformis, most frequently from 
the chest contents. 
Gram negative spirilla : Spirillum cholerae is found in the 
feces and is the only common gram negative spirillum. 
Gram Positive Organisms. 
Gram Positive Cocci: 
Staphylococci 
Streptococci 
Pneumococci 
M. tetragenus 
Gram Positive Bacilli: 
B. diphtheriae 
B. tetani 
B. tuberculosis 
B. anthracis 
These organisms may again be subdivided according to their 
location. 
Cerebro Spinal Fluid: 
Micrococcus intracellularis 
Pneumococcus 
B. tuberculosis 
Streptococcus 
B. influenzae 
Pericardial and Pleural Fluids: 
Pneumococcus 
Streptococcus 
B. mucosus capsulatus 
B. influenzae 
B. tuberculosis 
Peritoneal Fluids: 
B. coli 
Streptococcus 
B. tuberculosis 
Respiratory Tract: 
B. diphtheriae 
B. influenzae 
Streptococcus 
Pneumococcus 
B. mucosus capsulatus 
B. tuberculosis 
Micrococcus catarrhalis 
Actnomyces 
24 Feces: 
B. coli 
B. typhosus 
B. dysenteriae 
Gram positive anaerobes 
Many forms whose importance has not been worked out. 
Urine: 
B. coli 
Streptococcus 
M. gonorrheae 
B. typhosus 
B. tuberculosis 
Pelvic Organs: 
M. gonorrheae 
Streptococcus 
B. tuberculosis 
Many other forms probably unimportant. 
Gram Negative Cocci. 
Micrococcus intracellularis. 
Pathogenicity-This organism causes meningitis or spotted 
fever. 
Form-Small cocci usually appearing in pairs. The opposing 
sides are slightly flattened. They may be within leukocytes. 
Properties-They ferment sugars with acid production. 
Growth-They are rather difficult to culture at best. How- 
ever, we find that they grow best on Loeffler's medium. The 
colonies are white, shining and viscid. They tend to coalesce. 
The cultures must be transplanted every day or two in order to 
keep the organisms alive. 
Diagnosis-A lumbar puncture is made and the fluid obtained 
is centrifuged in a sterile tube. The sediment is then examined, 
microscopically, and also cultured on Loeffler's medium. 
Micrococcus Catarrhalis. 
Pathogenicity-This organism is of doubtful pathogenicity. 
Form-Similar in form to micrococcus intracellularis. It 
usually appears as a small diplococcus, but is not found intra- 
cellular. 
Properties-It does not ferment sugars. 
Growth-Small, round, greyish colonies with serrated edges. 
It cultures easily on agar and other ordinary media. 
Habitat--It is found in the mucous secretions of healthy 
persons. 
Micrococcus Gonorrheae. 
Pathogenicity-The cause of gonorrheae in man. 
Form-Small kidney shaped cocci usually appearing in pairs 
with their concave sides in opposition. They appear very much 
as a coffee bean. 
Properties-They do not ferment sugars or grow on ordinary 
media. 
25 Growth-They grow best on aseitie agar or blood serum. 
The colonies are small and yellowish white in color. The organism 
requires frequent transplantation. They are very difficult to 
culture. 
Diagnosis-The diagnosis is made from gonorrheal pus. The 
organism is found intracellular after the first few days. 
Gram Negative Bacilli. 
Bacillus coli. 
Pathogenicity-Non-pathogenic except in conditions of 
lowered vitality. Pyogenic when organs other than the intestinal 
tract are invaded. 
Form-Short motile rods which may be slightly curved. 
Properties-They are facultative anaerobic and have a rather 
sluggish motility. They coagulate milk rapidly, producing an 
acid reaction. 
Growth-They grow well on all media and form greyish 
undulated colonies on agar. 
Diagnosis-They may be found in the feces, urine, peritoneal 
fluid and in the pelvic organs. Their normal habitat is the 
intestinal tract. 
Bacillus Typhosus. 
Pathogenicity-This organism is the cause of typhoid fever. 
Form-A slender rod. 
Properties-It does not ferment sugars and will not coagidate 
milk nor produce acid. It is very motile. 
Growth-Scant growth on agar. It appears brownish yellow 
on gelatine and produces no change in litmus milk. 
Diagnosis-The organisms must be differentiated from bac- 
illus coli. This is best done by making a hanging drop prepara- 
tion and by culturing them on agar, gelatine, litmus milk and 
special sugar media. 
Bacillus Dysenteriae. 
This organism is practically identical with the bacillus 
typhosus both in appearance and in cultural characteristics, but 
it is non motile. When there is any doubt the clinical history 
must be differentiating feature. 
Bacillus Pyocyaneus. 
Pathogenicity-It is frequently the cause of suppuration and 
purulent middle ear inflammations. 
Form-It is a slender rod shaped organism from 2 to 6 
microns long. It has rounded ends and may often be seen in 
short chains. 
Properties-It is a facultative anaerobic, highly motile 
organism. A green color is formed in the presence of oxygen 
at room temperature. It liquifies gelatin and coagulates milk 
with acid production. 
Growth-It grows well on all media, appearing as large flat 
colonies with a greenish phosphorescence surrounding them. 
26 Diagnosis-The presence of the organism may be easily 
diagnosed from the greenish color of the pus. 
Bacillus Influenzae. 
Pathogenicity-This organism is the cause of epidemic in- 
fluenza. 
Form-Very minute rods usually clumped. 
Growth-Requires agar, which contains fresh blood. Growth 
is in the form of small, clear, almost transparent colonies. 
Diagnosis-They are easily found in the sputum of persons 
suffering from epidemic influenza. 
Bacillus Fusiformis. 
Pathogenicity-When associated with Spirochaetae they are 
considered the cause of ulcero-membranous angina and stomatitis. 
Form-They appear as long slender rods usually singly. 
Properties-They grow best under anaerobic conditions, pro- 
ducing a characteristic foul odor but no gas. 
Growth-In liquid media they form a flocculent white pre- 
cipitate. 
Diagnosis-No difficulty will be experienced in diagnosing 
them from all other organisms both culturally and micro- 
scopically. 
Gram Positive Cocci. 
Staphylococci. There are three types-aureus, al bus and citreus. 
Pathogenicity-All three of these types cause abscesses and 
suppurations. They are listed according to their respective 
virulence. 
Properties-They are aerobic, non-motile organisms which 
resist heat and drying as well as antiseptics. 
Form--These organisms appear in clusters like bunches of 
grapes. 
Growth-They grow well on all media, producing large round 
colonies. The color varies with the type of the organism. S. 
aureus develops a golden color, S. albus a white color and S. 
citreus a lemon yellow color. 
Diagnosis-Microscopically they are easily diagnosed. When 
cultured on agar their type is easily recognized by the color. 
Streptococci. 
Pathogenicity-The streptococci are high virulent pus pro- 
ducing organisms causing local suppurative processes, septicemia 
pyemia, erysipelas, etc. 
Form-They appear as chains of cocci. The individual cocci, 
which make up a chain, may vary greatly among themselves. 
Properties-They produce lactic acid in milk. They are 
facultative anaerobes. 
Growth-On agar they appear as small, very transparent 
drops having a slightly bluish tint. They culture on Loeffler's 
medium. 
Diagnosis-One glance at a typical field assures one of 
streptococci. 
27 Pneumococci. 
Pathogenicity-They are the casual organism in pneumonia 
and other respiratory troubles. 
Form-They appear as large, lancet shaped cocci, occurring 
singly or in pairs or short chains. In their natural environment 
they have a well marked capsule surrounding each coccus. 
Properties-They do not liquify gelatine or produce acid. 
Growth-They grow well on Loeffler's serum medium. When 
cultured artificially, they do not produce a capsule. 
Diagnosis-Their shape, staining reaction and cultural char- 
acteristics, together with the capsule, which appears as a halo 
in the unstained specimen, make diagnosis easy. 
Micrococcus Tetragenus. 
Pathogenicity-Th:s organism is found in 80% of all cases 
of cavernous phthisis. It is not known to be pathogenic to man. 
When found in the sputum it is well, however, to examine the 
patient for tuberculosis. 
Form-They appear in groups of four, and when growing in 
the body have a well marked heavy capsule surrounding them. 
Properties-Gelatine is not liquified and milk is not coagu- 
lated by this organism. 
Growth-On agar a rough white confluent growth results. 
It grows well on all media. 
Diagnosis-The typical microscopic appearance will differen- 
tiate this organism from all others. The deeply staining cocci 
are grouped in a square, surrounded by a thick homogeneous 
capsule, thus giving the group a round appearance. They cannot 
be mistaken or confused. 
Gram Positive Bacilli. 
Bacillus diptheriae. 
This organism is identified by its arrangement and morph- 
ology to be considered later. 
Bacillus Tetani. 
Pathogenicity-This organism is the cause of tetanus. 
Form-It is a long, slender, rod shaped bacillus, the spore 
forming stage of which has a drum stick shape. It may occur 
in long chains or threads. 
Properties-It is a strictly anaerobic organism and, there- 
fore, is not found in the superficial parts of wounds. It grows 
slowly on gelatine, liquifying the medium. The spores are formed 
after 24 to 56 hours at incubator temperature (37% degrees C.). 
In agar stab cultures the growth resembles a fir tree with 
delicate branches radiating from a central core. Blood serum 
medium is liquefied. Milk is coagulated with the production of 
acid. 
Diagnosis-In taking specimens for diagnosis procure the 
material from the deep parts of the wound. Make a stab culture 
of this material on agar, i. e., instead of spreading the material 
on the surface of the medium push the platinum needle through 
the surface and down almost to the bottom of the tube. Incubate 
28 24 to 36 hours. Look for the characteristic growth. Examine 
both the growth and specimen microscopically for the organism. 
Bacillus Tuberculosis. 
Pathogenicity-This organism is the cause of all tuberculous 
lesions. 
Form-Bacillus tuberculosis appears as short, slender rods, 
which are often slightly curved and may appear either singly or 
in pairs or groups. 
Properties-It is very difficult to start a culture of bacillus 
tuberculosis from a lesion even on the most favorable media. 
After one has been started, the organism grows well on egg media, 
glycerine agar, etc. For starting the culture the use of Dorset's 
egg medium is recommended. The colonies appear after several 
days incubation as very minute white grains. They are dull and 
dry in appearance and have an irregular outline. After the 
culture has been transplanted several times, it grows much more 
luxuriently. The surface of the medium may be covered with a 
dull grayish or yellowish white film. This organism belongs to 
the acid fast group; that is, when it is stained with steaming hot 
carbol fuchsin, it is not bleached with acid. The staining method 
is as follows: 
1. Cover the smear with carbol-fuchsin stain. Hold above 
the flame, permitting it to steam, not boil, for 3 to 5 minutes. 
2. Pour off the stain and wash with water. 
3. Wash in 95% ethyl alcohol containing 2% hydrochloric 
acid until no more stain is given off. 
4. Wash with water. 
5. Counter stain with methylene blue 30 seconds. 
6. Wash in water, dry, mount in balsam and examine. 
The tubercle bacilli appear red while all other stains blue. 
Diagnosis-Bacillus tuberculosis may be found, in spinal, 
peritoneal, pericardial and pleural fluid; also in the respiratory 
tract, urine and the pelvic organs. The fluids should be centri- 
fuged and the precipitates stained by the acid fast method. 
Bacillus Diphtheriae. 
Pathogenicity-This organism is the cause of diphtheria. 
Form-It appears in the form of non-motile rods of varying 
length, size and shape, depending upon the age of the culture 
and the media, on which they have been grown. On Loeffler's 
medium, in a 6 to 10 hours' culture, we find straight or slightly 
curved bacilli either club shaped or pointed at both ends. In 
older cultures, lancet, dumb-bell and spindle forms are seen. In 
these bacilli we find small highly refractile granules. 
Method of procedure in making a throat culture: For ex- 
amination in a case of suspected diphtheria, wrap a piece of sterile 
cotton around a wooden applicator. Carefully swab the throat 
and tonsils of the suspected individual. Then, with the usual 
sterile precautions inoculate the surface of the medium, (Loeffler's 
medium should be used), with the suspected material obtained 
from the patient's throat. This is accomplished by passing the 
29 inoculated swab over the surface of the medium. Caution: Be 
sure to destroy the swab after using. 
Growth-The organism grows most rapidly on Loeffler's 
medium, forming small or large, round, greyish white, granular 
colonies, thick in the center and thin at the edges. 
B. Diphtheriae is characterized by its arrangement, occurring 
in clumps, in which the rods lie side by side, and by the presence 
of deeply staining granules or faintly staining bars. These char- 
acteristics are best seen in culture 14 to 24 hours old. Make a 
thin smear in the usual way from the growth on the surface of 
the medium. Fix it in the flame and stain with methylene blue 
in the usual manner. Wash in water, mount the cover slip in 
water and examine with the oil immersion objective. If granules 
and bars are not recognizable, place a drop or two of .3% acetic 
acid at the edge of the cover slip and draw it through by filter 
paper held to the opposite edge. Granules and bars, if present, 
stand out very distinctly by this method. Usually each organism 
has two granules-one at either end. Some, however, have a 
third in the center. 
Bacillus Anthracis. 
Pathogenicity-The bacillus of anthrax is the cause of splenic 
fever, or anthrax in animals and malignant pustule in man. 
Form-These organisms are large rod shaped bacilli, occur- 
ring in long threads. They have a capsule and form spores under 
aerobic conditions. The ends of the organism appear to be 
square. 
Growth-Bacillus anthracis grows well on all media. On 
agar the colonies are greyish white and appear to be slightly 
elevated above the surface of the medium. When it is viewed 
under low power the colony appears like a tangle of hair. Gelatine 
is liquefied and milk is coagulated with acid production by this 
bacillus. 
Diagnosis-The organism is usually easily recognized by its 
size and staining reactions, in smears made from the fluid of the 
blebs and vesicles present about the lesion. 
Entamoeba Buccalis. 
Pathogenicity-Entamoeba buccalis is considered by some 
the direct cause of pyorrhea. 
Form-Entamoeba buccalis is a large organism of varying 
form. It moves by extending one part and gradually flowing 
towards it. In this mass of protoplasm there are granules, the 
granular protoplasm being surrounded by a layer of hyalin ecto- 
plasm. Near the center of the cell is a nucleus. 
Habitat-The entamoeba is not known to live or reproduce 
except in the mouth and adjacent structures in man. It may be 
found in large numbers in the pus from pyorrhea pockets. 
Method of examination-These organisms, while they may be 
stained, are usually examined alive, because unless movement is 
seen, it is very difficult to differentiate them from swollen 
epithelial cells, etc. Entamoeba buccalis very quickly loses its 
30 movement, if not kept at body temperature and, therefore, an 
electrically heated warm stage is used to keep the preparation as 
near 37% degrees as possible. The specimen is best observed in 
a hanging drop preparation made in the following manner: 
Electrically Heated "Warm Stage" 
1. Clean a concave slide and a cover slip. 
2. With a toothpick or small brush place a little vaseline 
around the edge of the cover glass. 
3. Place a droplet of normal salt solution in the center of 
the cover. 
4. With a platinum loop make an emulsion of a little of 
the suspected material and the drop of normal salt solution. 
5. Turn the slide upside down and place on top of the cover 
so the drop is in the center of the concavity. 
31 6. Turn the slide right side up quickly, so the drop will 
still remain in the center of the cell. The preparation is now 
ready for examination. 
Method of observation-Place the warm stage on the micro- 
scope, connect it and turn on the current. After allowing a few 
minutes for it to heat, place a freshly made preparation on the 
stage. Examine first under low power and then with the high 
power objective. Cut down the light with the diaphragm until 
a satisfactory illumination is obtained. This is very important in 
examining all unstained preparations. 
Appearance of the entamoeba-They appear as large ir- 
regular masses of protoplasm distinguished by their motility. 
They seem to extend themselves and flow along. Occasionally 
their movement will be very sluggish. If a rough sketch of them 
is made and then compared with their shape after a few minutes, 
identification may be made easier. 
Diagnosis-The material for diagnosis is obtained by press- 
ing out the contents of pus pockets and from the material around 
the base of the teeth. 
Entamoeba Histolytica. 
Pathogenicity-Entamoeba histolytica is considered the cause 
of amebic dysentery. 
Form-This organism appears as a rather large, irregular 
mass of protoplasm, 30 to 50 microns in diameter, and is very 
motile. In its cytoplasm may be seen red corpuscles, bacteria, 
etc. This condition is never seen in nonpathogenic amebae of 
the intestinal tract. The differentiation into a granular entoplasm 
and a hyaline ectoplasm is well marked in this species of ameba. 
Habitat-Entamoeba histolytica is found in the intestinal 
tract of persons suffering from amebic or tropical dysentery. It 
may be passed out in the blood streaked mucus or in blood clots 
together with the feces. Musgrave and Clegg advocate the ad- 
ministration of a saline cathartic, which would tend to flush out 
the colon, and examination of the liquid parts of the stool. The 
stool should not be allowed to become cool, as the amebae quickly 
lose their motility. 
Method of examining-In selecting the particles for examina- 
tion, the most likely are the small, blood streaked mucous shreds 
or the fluid in their vicinity. A little of this material is mixed 
with a drop of normal saline on a cover glass and is prepared and 
examined by the method outlined for examining entamoeba 
buccalis. 
Widal Test. 
Taking the Blood-Make a puncture as for a blood count 
after using the usual sterile precautions and collect the blood 
in a capsule, made by drawing out a short piece of glass tubing 
into a capillary point at each end. Allow the blood to stand until 
the serum separates from the clot or centrifuge to obtain the 
serum. 
32 Dilution of the Serum-Take a little of the serum in a pipette. 
Place one drop in a small test tube or watch glass. Now add 19 
drops of sterile normal salt solution. This makes a dilution of 
1 to 20. If whole blood is used, it may be collected directly in 
the white pipette of the hemacytometer outfit. In this case make 
a puncture in the usual way and, when a large, fresh drop of 
blood has collected, immerse the tip of the white pipette marked 
No. 11 in it. Draw the blood up to the mark 1 on the stem. 
Dilute at once with sterile normal salt solution. This makes a 
dilution of one part of serum to forty parts of salt solution (the 
blood is half serum). 
In cases where it is impossible to do this test at once, dried 
blood may be used. Place a large drop of blood on a glass slide. 
Permit it to dry. When ready to make the test, scrape off the 
blood, place it in a watch glass and add 9 drops of salt solution. 
This makes a dilution of 1 to 20. Dried blood is half serum. 
Preparing the actual test-Clean a hollow ground slide and a 
cover slip. Grease the edge of the cover slip with a little vaseline. 
Take one loopful of the bouillon culture of Bacillus typhosus. 
Put it in the center of the cover slip. Sterilize the platinum loop 
and take a loopful of the 1 to 20 serum. Mix the two. Place the 
inverted slide over the cover slip, so that the drop is in the center 
of the concavity of the slide. Press the slide down. Invert the 
preparation. Place it on the stage of the microscope. Observe 
the reaction, using the high dry lens and a subdued light. The 
organisms, when first noted, will be seen to be actively motile. 
They lose this motility and clump in groups of five or more bacilli, 
when the test is positive. This clumping and loss of motility of 
a majority of the organisms, occurring within 45 minutes, is con- 
sidered a positive Widal test. The culture referred to in the last 
paragraph should be a fresh culture in bouillon of Bacillus 
typhosus. A culture of Bacillus typhosus on agar should always 
be kept in stock. An old laboratory culture, which has been 
transplanted many times, is often more 'motile and active than a 
freshly isolated one. A stock culture may be kept at room tem- 
perature and transplanted every three or four weeks. 
Before making the test inoculate a tube of bouillon with a 
little of the stock culture. After from 18 to 24 hours at incubator 
temperatures the organisms will be ready. It is well to take a 
loop of the emulsion and one of normal salt solution and put them 
on the slide and examine to see, that the organisms are motile 
and not spontaneously clumped. 
The India Ink Method of Finding Treponema Pallidum. 
Scrape the surface of the suspected lesion lightly with a 
shai p scalpel. If the ulcer seems to contain the typical soft, 
gummy substance, the scraping may be omitted. 
Drop a few, 2 or 3, drops of warm normal saline solution on 
the surface of the lesion. Quickly withdraw it again, using a 
nipple pipette. 
33 Place a droplet of some high grade india ink on a slide. To 
this add 1 or 2 small drops of the suspension. Mix the two with 
a platinum wire. Be careful not to spread the smear too much. 
Permit the preparation to dry. Search for a field where the 
organisms appear pearly against a grey or black background. 
Cracks, cotton fibers, etc., should not be confused with the true 
treponema. This method has the advantage of being rapid and 
simple. The true treponemata appear as fine, closely convoluted 
spirals, of from 6 to 40 turns. The organism tapers at the ends. 
Other organisms, which may simulate the organism of syphilis, 
must be borne in mind and must be excluded, before a diagnosis is 
made. It is useless to search for the organism, when the patient 
is undergoing rigorous treatment. 
34 HI.-SPUTUM EXAMINATION 
Sputum is that material brought up from the pharynx and 
respiratory tract by the act of coughing. In pathological condi- 
tions the quantity may vary from a little to a great deal. 
General Characteristics. 
In sputum examination note is made of color, consistency 
and odor. 
Color. 
1. Bright red and foamy; may be due to hemorrhage from 
eroded blood vessels. 
2. Current jelly; hemoptysis resulting from pulmonary 
neoplasms. 
3. Prune jelly; inflammatory edema, complicated with 
pneumonia. 
4. Rusty; Pathognomonic in' pneumonia. 
5. Yellowish red liver abscess, rupturing into the lung. 
6. Yellowish green, coinlike lumps; influenza. 
7. Greenish brown ; pulmonary gangrene. 
8. Green; pulmonary infection, complicated by jaundice. 
Bacillus pyocyaneus and other organisms may stain the sputum 
with their products. 
Consistency. 
Mucoid sputum is greyish white in color and is tough and 
thready in consistency. 
MUCO-PURULENT SPUTUM-Is near homogeneous, non- 
transparent and yellowish white in appearance. It is of a sticky 
or gluey consistency. In a good light the mucoid portions may 
be clearly distinguished from the purulent parts. 
PURULENT SPUTUM-Is a thick liquid in consistency, hav- 
ing a characteristic appearance and varying from a yellowish 
white to a greenish white in color. 
SANGUINEOUS SPUTUM-Blood, when coming from the 
lungs, is usually bright red in color and foamy. If it has coagu- 
lated before being expectorated, it may form clumps and clots 
of varying color according to its age. 
SERO-SANGUINEOUS SPUTUM-Is commonly called prune 
sputum because of its color and consistency. 
PURULO-SANGUINEOUS-Is an admixture of blood and 
pus. It occurs in large cavities in the lungs. If this material is 
expectorated soon after it is discharged, it appears in the form 
of clumps or clots, which are stained bright red. When large 
amounts of pus are present, this may be a dirty greyish red. If 
the material is not expectorated for a considerable time, after it 
is discharged, it has a homogeneous appearance and a dirty red 
to a brownish-grey appearance. 
35 Odor-Freshly expectorated sputum as a rule has no char- 
acteristic odor. When sputum has a foul odor, it is due to decom- 
position, either before or after bein expectorated. If this occurs 
in the body the sputum often has a foul, repulsive odor as in 
fetid bronchitis, pulmonary gangrene, etc. The sputum in certain 
diseases may have a sweetish odor, as in perforating empyema 
and certain ulcerative processes. It may also have an old cheese 
odor characteristic of some cases of empyema. 
Special Characteristics. 
Dittrich's plugs-These are greyish white particles, varying 
in size, and found in the sputum in conditions of pulmonary 
gangrene. They are composed chiefly of debris and are rich in 
micro-organisms. 
Curschmann's spirals-These are spiral threads of mucus, 
which may be covered with leucocytes. They are easily seen with 
low magnification and are found in bronchial asthma. 
Fibrin casts-These are coagulated fibrin casts of the bronchi, 
which are expectorated in the sputum. They may be several 
centimeters long and appear as white or greyish white, homogen- 
eous, cylindrical, branching structures. 
Elastic fibres-These fibres are found in purulent portions of 
the sputum. They appear as highly refractile, waxy, double 
contoured fibres. 
Charcot-leyden crystals-These crystals are clear, pointed 
and octahedral in shape. They are usually present in large num- 
bers, when the sputum has been exposed to the air for a short 
time. They are found especially in asthmatic conditions. 
Cells Found in the Sputum. 
Pus cells-These are easily recognized by their size, shape, 
nuclei, etc. 
Red blood corpuscles-The red cells are recognizable as 
orange biconcave disks. 
Squamous cells-These are large, flat, irregularly shaped 
cells. They come from the mouth, pharynx or vocal cords. 
Columnar cells-These may be ciliated. They come from the 
nose, larnyx or bronchi. 
Alveolar epithelial cells-These are round, oval or irregularly 
shaped cells. As a ride they have undergone degeneration, when 
found in the sputum. 
Heart disease corpuscles-This is the name given alveolar 
epithelial cells, which contain blood pigments. They appear in 
conditions of chronic congestion of the lungs caused by cardiac 
insufficiency. 
Method of Obtaining Sputum. 
When collecting sputum for examination the morning sputum 
or the entire amount for 24 hours should be saved. The morning 
sputum in early stages of tuberculosis may contain tubercle 
bacilli, when they cannot be detected in sputum at any other 
part of the day. The patient should be instructed to rinse the 
36 mouth well before expectorating, in order to prevent contamina- 
tion by food particles and also by bacteria and other material in 
the mouth. Only the sputum, which actually comes from the 
lungs and bronchi, should be saved. Material from the upper air 
passages can be identified by its heavy mucous character and by 
the type of epithelial cells. 
Outline for Routine Examination. 
1. Spread the sputum in a thin layer in a petri dish and 
place it on a black background. 
2. Examine all parts with a hand lens and select the parts 
most suitable for microscopic examination. 
3. Transfer all interesting and suspicious particles to clean 
slides, cover and examine unstained with the microscope. (In 
examining unstained preparations a subdued light gives the best 
results.) 
4. After examining the smears slip the covers from those 
you may wish to stain. This leaves a good smear on both slide 
and cover slip. 
5. Air dry the smear. Fix in the flame and stain by one 
of the following methods: 
a. For cells and bacteria-methylene blue. 
b. For Bacillus tuberculosis-the carbol-fuchsin method 
given earlier. 
c. Gram's method for differential diagnosis of bacteria. 
Organism Commonly Found in the Sputum. 
Bacillus influenzae 
Bacillus mucosus capsulatus 
Bacillus pyocyaneus 
Micrococcus catarrhalis 
Gram negative 
Gram positive 
Pneumococci 
Bacillus tuberculosis acid fast 
Staplylococci 
Streptococci 
(See chapter on Micro-organisms for descriptions.) 
After examination is completed destroy the sputum either 
by heat or some powerful germicide. Sterilize everything which 
has come in contact with it. Guard against dry dissemination of 
infective material. Do not permit the presence of flies in your 
laboratory. 
37 IV. BLOOD 
Apparatus. 
Microscope 
Hemocytometer 
Tallquist hemoglobin scale 
Blood lancet 
Slides 
Cover glasses 
Reagents. 
0.3% acetic acid 
Wright's stain 
Hayem's solution: 
Mercuric chloride 0.5 gm. 
Sodium chloride 1.0 gm. 
Sodium sulphate 5.0 gm. 
Distilled water 200.0 c c. 
A clinical examination of the blood should include the follow- 
ing points: 
1. The estimation of hemoglobin. 
2. A count of the red blood corpuscles. 
3. A count of the white blood corpuscles. 
4. Color index. 
5. An examination of a stained film. 
Normal Blood. 
Normal blood contains the following: 
1. Erythrocytes. 
1. Erythrocytes are normal red cells, 7.5 microns in 
diameter. 
2. Microcytes--undersized red cells, a few are some- 
times found in normal blood. 
3. Macrocytes-oversized red cells, occasionally found 
in normal blood. 
2. Lymphocytes. 
1. Lymphocytes-small, form 15 to 30% of the whites. 
Single, large, oval or round mucleus nearly filling the 
cell, clear cytoplasm. 
2. Large lymphocytes-3 to 10% of the whites. Larger 
than the small lymphocytes. 
3. Leucocytes-Polymorphonuclear leucocytes. Slight- 
ly larger than red blood corpuscles. The nucleus is 
lobulated or may appear to be made up of several 
distinct parts. According to the staining reaction of 
«■ the granules of the cytoplasm they may be classed as 
neutrophiles, eosinophiles and basophiles. 
a. Neutrophiles-60 to 70% of the white corpuscles. 
The nucleus is either lobated or divided into 
several separate parts. The cytoplasm is filled 
with fine lilac staining granules. 
38 b. Eosinophiles-1 to 4%. They have large eosin 
or acid staining grannies. The nucleus is either 
lobated or separated. 
c. Basophiles-1%. They have large blue staining- 
granules and a nucleus, which is either lobated 
or divided. 
4. Transitional leucocytes. The cytoplasm in these is 
faintly granular. The nucleus is not perfectly round, 
but appears to be slightly kidney shaped. 
Pathologic Cells Found in the Blood. 
Abnormal red cells. 
1. Microcytes-4 to 5 microns in diameter. They are ab- 
normally small non-nucleated red cells. 
2. Macrocytes-8 to 10 microns in diameter. Abnormally 
large non-nucleated red cells, usually found after acute 
hemmorrhage. 
3. Normoblasts-These are nucleated red cells whose necleus 
is round and deep staining. They are usually about the 
size of normal red corpuscles. 
4. Megaloblasts-10 to 14 microns in diameter. These are 
large cells with a round, granular nucleus, not deeply 
stained. 
5. Poikilocytes-These are irregularly shaped non-nucleated 
red cells. They may appear comma shaped. They should 
not be confused with crenated cells. 
Abnormal White Cells. 
Myelocytes-These are large non-nuclear white cells which 
have round, moderately deeply stained nuclei, situated excentri- 
cally. The cytoplasm contains granules which are either euetro- 
philic, i. e., they take a lilac color when stained with Wright's 
stain; or eosinophilic, taking a red stain under the same condi- 
tions ; or basophilic. 
Hemoglobin Content 
Hemoglobin content-Hemoglobin is the active substance in 
the red cells. It has the power to form a loose combination with 
oxygen, thus transporting it and exchanging it, for carbon dioxide 
in the vascularized tissues of the body. The hemoglobin content 
of the blood is a fairly constant factor in health. Tn certain 
diseases it may be diminished although the number of red cor- 
puscles is normal; this is called oligochromemia. While in others 
it may be reduced due to decrease in the number of the red cells. 
This is called oligocythemia. The hemoglobin is estimated by 
means of Tallquist color chart. Instructions accompany each 
chart. The color chart is a series of colors representing definite 
percentages of hemoglobin. A book of white filter paper accom- 
panies each chart. A drop of blood is taken from the ball of the 
finger or lobe of the ear and placed on the white filter paper. 
It is then compared with the color scale, the blood is matched and 
the percentage may be read directly from the scale. This 
method is easy, rapid and accurate enough for clinical work. 
39 The Enumeration of Red Blood Corpuscles. 
The corpuscles are counted with the aid of a hemocytometer. 
This outfit consists of two special graduated pipettes, a special 
slide and a ground cover glass. 
0.100mm. 
Jo(lmlrn 
C. Zeiss 
Jena 
Hemocytometer 
The pipettes are graduated capillary tubes with a mixing 
chamber at the top of each. They have rubber tubes with 
mouthpiece attached to them. One of these pipettes is graduated 
for 1% solution. It is used for counting the red cells. The 
other is graduated for a 10% solution. This is used for counting 
the white cells. The upper part of each pipette is a mixing 
chamber in which the blood is mixed with a diluting fluid. 
Method-Sponge the site of the puncture with alcohol. Then 
prick it with a sharp scalpel or lancet. Wipe away the first 
drop of blood. When a large, fresh drop collects immerse the 
tip of the pipette marked 101 and draw the blood up to the mark 
1 on the capillary tube. Then dilute the blood by drawing up 
Hayem's solution to the mark 101. Move the tube while mixing 
the diluting fluid with blood as it comes in. Shake the pipette, 
keeping both ends closed until the blood is thoroughly mixed 
with the diluting fluid. If you desire to transport the specimen 
to your laboratory, remove the rubber tube and place a wide 
rubber band around both ends of the tube, thus satisfactorilly 
closing them and preventing leakage. 
40 Counting the cells-Have the slide and cover slip clean. 
Blow out the first drop from the pipette, as it is only diluting 
fluid. Then put a small droplet on the "land" (the elevated 
part in the center of the slide). Place the cover on with care, 
trying not to have a bubble in the field to be observed. Press 
down the cover. Note Newton's color rings. They should appear 
in all good preparations. Place the preparation aside for a few 
minutes to permit the corpuscles to settle. Count under high 
dry lens. The central part of the ruling is used for counting 
Tuerk Ruling 
red cells. Focus the microscope of the center of the land. When 
the preparation is in the focus the red cells will be seen in the 
squares which are ruled on the land of the slide. The large 
central square is divided into 16 smaller ones. Each small square 
is divided into 16 still smaller ones. Each of these smaller squares 
represents 1/4000 of a cubic millimeter of diluted blood. 
Count all the cells in each of the small squares and also those 
touching the upper and left hand sides of the square, but not 
those that touch the other sides. For an accurate count it is 
advisable to count 5 large squares, i. e., 80 small ones. 
The count is computed by the following method: 
X equals RxDx4000 
s 
R equals the number of red cells counted. 
1) equals the dilution, usually 100 times. 
S equals the number of small squares. 
Example: 
1280 equals the number of red cells counted. 
100 equals the dilution. 
80 equals the number of squares counted. 
1280x 100x400 , q 
equals 6,400,000 red cells per cubic 
$0 millimeter. 
The Enumeration of White Cells. 
The leucocytes are counted in a similar way to that used in 
counting the red cells. 
41 The blood is taken in exactly the same way, except that the 
pipette marked No. 11 is used. This pipette gives a dilution of 
1 to 10. A solution of 0.3 to 0.5% glacial acetic acid tinted with 
methylene blue is used as a diluent in place of the Hayem's 
solution, used in counting red corpuscles. 
Because of the smaller number of white cells the corners 
of the rulings are used. 16 of these large squares make up a 
corner, and each one is equal to 16 of the smaller squares. Each 
of the larger squares is equal to 1/250 cu. m.m. For accurate 
results it is advisable to count 50 large squares. Count all the 
cells in the squares and those touching the upper and left hand 
sides of the squares. The computation is as follows: 
X equals Wx I) x 250 
S 
X equals number of leucocytes per cubic millimeter of blood. 
W equals number of leucocytes counted. 
S equals number of squares counted. 
Example: 
50 squares counted Dilution is 10. 
160 leucocytes seen. 
160x10x250 , Qnnn , , i- r 
equals 8,000 leucocytes per cubic centi- 
50 meter. 
After finishing a count blow out the contents of the pipette. 
Wash it out with water and fill it, shaking and then blowing the 
contents out. Do the same with alcohol and ether. Be sure the 
glass ball in the mixing chamber rolls freely and that the capillary 
tube is clean before putting the instrument away. In case a 
little blood cannot be washed out it may be digested out with 
pepsin and 0.5% hydrochloric acid. 
Color Index. 
This is important, since it often aids in making a diagnosis. 
The color index is the relation between the percentage of hemo- 
globin and the percentage of red cells, 5,000,000 red cells being 
considered normal. 
Method. Examples: 
The estimation of hemoglobin is 50%. 
The red cell count is 5,000,000, i. e., 100%. 
100 i r i i-i 
-777 equals .5, a low color index. 
50 
Second case: 
Estimation of hemoglobin, 50%. 
Number of red cells, 2,500,000, i. e., 50%. 
50 , , , , - n 
equals 1, a normal color index. 
50 
Third case: 
Estimation of hemoglobin, 80%. 
Number of red cells, 2,000,000, i. e., 40%. 
80 
-^equals 2, a high color index. 
42 Microscopic Examination of the Blood. 
Method of smearing-The slides should be carefully cleaned. 
Make a puncture in the usual way. Take a fresh drop of blood 
on the end of a slide. Touch the end of a second clean slide 
to the drop of blood, holding the two slides so that an acute angle 
is formed between them. Now draw the upper slide over the 
surface of the lower and a good smear should result. Make 
several of these. Permit them to dry in the air. If necessary 
they may be wrapped in paper and transported to the office for 
staining and examination. 
Staining the smear-Wright's stain is the most satisfactory 
simple stain. It may be purchased ready for use in one-ounce 
bottles. Put a few drops of stain on the smear, spreading them 
so they will cover the entire smear. Count the number of drops 
used. Permit the stain to remain two minutes then add an equal 
quantity of distilled water. Oscillate the slide for three minutes. 
Wash in tap water for 30 seconds. The preparation should assume 
a rather pink color. Dry the preparation between filter paper 
and then in the air. Put a small drop of Canada balsam near the 
center of the preparation and then a clean No. 1 cover glass. 
Press the cover glass down. The preparation is now ready to be 
examined. This should be done with the high dry and oil im- 
mersion lenses. The examination may be made without a cover 
glass by placing a drop of immersion oil directly on the stained 
and dried smear. 
The cells appear as described on the first page of this chapter. 
43 Differential Diagnosis of Blood Diseases. 
SECONDARY ANAEMIA-Hemoglobin reduced, red cell 
count lowered, color index lowered. Cells usually found: micro- 
cytes, macrocytes, porkilocytes. 
Stained Film-Normoblasts and very rarely megaloblasts, 
qualitative changes only in severe cases. 
CHLOROSIS-Clinical findings important. Hemoglobin low. 
Red cell count slightly reduced, may be normal or high. Color 
index very low. 
Stained Film-Red cells show lack of hemoglobin, normo- 
blasts may be found. There is a tendency to relative lymph- 
ocytosis. 
PERNICIOUS ANAEMIA-Blood pale and watery, coagu- 
lates slowly. Hemoglobin low, 20 to 40 per cent common. Red 
cell count very low, one to two million common. Color index 
high, may be 1 to 1.5 (the only condition in which the color index 
is high). 
Stained Film-Cells scattered, porkilocytes, more mycrocytes 
than macrocytes, megalo blasts exceed normo blasts. Leupaenia 
may be only 3000 to 4000 white cells. 
SPLENO-MYOLOGENIOUS LEUKAEMIA-Hemoglobin re- 
duced as the disease progresses. Red cells greatly reduced with 
the advancement of the disease. Color index low. Enormous 
increase in leucocytes, 300,000 common. 
Stained Film--Mylocytes about 30%, blasts common and 
often more numerous than in anaemia of a like grade due to other 
causes. 
LYMPHATIC LEUKAEMIA-Hemagiobin low, red cell 
count low, color index low, white cell count very high but not as 
high as in the myelogenous type. 
Stained Film-Nucleated red cells may be present but in 
smaller numbers than in the myologenious leukaemia. The dif- 
ferential count shows 80 to 90 per cent of the white cells to be 
lymphocytes. 
44 A Well Appointed Laboratory Table 
45 V.-SPINAL FLUID 
Apparatus. 
Microscope 
Hemacytometer 
Lumbar puncture needle 
Test tubes, 3 inch by % inch. 
Pipettes graduated in 0.1 cc. 
Slides 
Covers 
Reagents. 
Fluid for spinal count: 
Methyl violet 0.2 gram 
Glacial acetic acid 5.0 c c. 
Distilled water qsad 100.0 c c. 
10% Butyric acid (for Noguchi test) : 
Butyric acid 10.0 c c. 
Physiological salt solution qsad 100.0 c c. 
4% Sodium hydroxide solution (for Noguchi test). 
Saturated aqueous ammonium sulphate solution (for Ross- 
Jones test). 
Normal cerebrospinal fluid is clear, colorless, contains not 
over 10 cells per cu. m.m. and has a very low protein content. 
In pathological conditions involving the meninges, the fluid be- 
comes cloudy, due to an increased cell content and the protein 
content is increased. 
Cell count-Fill the hemacytometer pipette marked 11 to 
the mark 1 with the methyl violet solution and then fill to the 
mark 11 with uncentrifuged cerebrospinal fluid. Shake thor- 
oughly; allow to stand for five minutes; discard the first drop 
blown from the pipette; place a drop of the mixed fluid on the 
ruled area of the hemacytometer counting chamber and adjust 
the cover glass. Count all the cells seen in the entire ruled area 
of sq. m.m., using the low power of the microscope. Multiply 
the number of cells counted by 11 and divide by 9. The result is 
the number of cells per cubic m.m. of cerebrospinal fluid. 
Noguchi test-To one part (0.1 cc.) of spinal fluid, add five 
parts (0.5 cc.) of 10% Butyric acid solution. Ileat to boiling. 
Add, immediately, one part (0.1 cc.) of 4% sodium hydroxide 
and boil again for a few seconds. A granular or flocculent 
precipitate indicates a positive reaction, i. e., an increased protein 
content. If the precipitate does not form at once, at least 20 
minutes should be allowed before the test is considered negative. 
Opalescence or turbidity without a definite precipitate is con- 
sidered a negative result. 
Ross-Jones test-Place 2 cc. of standard ammonium sulphate 
solution in a small test tube. Carefully layer 1 cc. of spinal fluid 
over this, by allowing the fluid to flow down the side of the in- 
clined tube. A greyish white ring forms within 3 minutes at 
the line of junction of the two fluids, if the test is positive. 
46 Bacteriological examination. The general directions given 
in the section on bacteriology are to be followed. If cultures 
are to be made, the material for inoculation should be taken before 
the fluid has been contaminated by the other examinations made. 
Microscopic examination of the sediment, obtained by centrifug- 
ing the fluid, gives valuable information. Stain smears with 
methylene blue and by the gram method. From the methylene 
blue preparation, a differential count giving the relative propor- 
tion of polynuclear leucocytes and lymphocytes can be made. If 
tuberculous meningitis is suspected, stain the smear by the 
tubercle bacillus method given in the section on bacteriology. 
47 VI.-STOMACH CONTENTS 
Apparatus. 
Stomach tube with bulb and funnel. 
4-inch funnel. 
4-inch porcelain evaporating dish. 
10 cc. pipette. 
25 cc. burette graduated in 0.1 cc. 
Test tubes, 6 inches. 
Reagents. 
Congo-red paper. 
1% Phenolphthalein in 95% alcohol. 
1% Alizarin S in water; filtered. 
Toepfer's reagent: 0.5% Di-methyLamido-azo-benzol in 95% 
alcohol. 
5% Ferric chloride in water. 
N/10 Sodium hydroxide. 
Qualitative Examination. 
After a test meal, the amount of fluid obtained should be 
measured and the odor, color and gross characteristics of the 
material should be noted. The presence of free hydrochloric acid 
is determined by the blue color, which results, when a piece of 
Congo-red paper is moistened with the fluid. 
Lactic acid-To a test tube of distilled water add a few 
drops of 5% ferric chloride solution until a very faint yellow 
color results. Divide the fluid into two parts and to one add a 
few drops of gastric fluid. Lactic acid produces a distinct canary 
yellow color in the tube to which it has been added. 
Blood-Use the guaiac test as described for urine. 
Boas-Oppler Bacillus-When the hydrochloric acid content is 
low, this organism may be present. When present at all, the 
bacilli are usually numerous. They are large rods often arranged 
in chains. They are actively motile in the fresh preparation and 
they stain by the gram method. 
Quantitative Examination. 
For quantitative examination, filter the fluid through two 
thicknesses of gauze in a funnel. 
Free hydrochloric acid-Measure 10 cc. of the filtered juice 
into a porcelain dish and add a drop of Toepfer's reagent. Fill 
a burette with N/10 sodium hydroxide and note the reading. 
Run in the sodium hydroxide from the burette until the orange 
red color changes to a permanent bright yellow. The result is 
expressed by the number of cc. of sodium hydroxide used multi- 
plied by 10. The normal figure is 20 to 60. 
Total acidity-The free hydrochloric acid having been de- 
termined, add to the same specimen a few drops of 1% phen- 
48 olphthalein solution. Continue the titration with N/10 sodium 
hydroxide until a permanent pink color results. The total num- 
ber of cc. of sodium hydroxide used, including that used for the 
determination of free hydrochloric acid multiplied by 10 gives 
the total acidity. The usual number is between 40 and 80. 
Combined hydrochloric acid. Measure 10 cc. of the filtered 
fluid into a porcelain dish and add a few drops of 1% aqueous 
alizarin solution. Add N/10 sodium hydroxide from the burette. 
The color changes from yellow to red and then to violet, which 
is the end reaction desired. Multiply the number of cc. of 
sodium hydroxide used by 10 and subtract from the figure ob- 
tained for the total acidity. The normal reading varies from 
0.5 to 15. 
From the three determinations made as above, the follow- 
ing calculations can be made: 
Combined hydrochloric acid plus free hydrochloric acid 
equals the total hydrochloric acid which is physiologically active. 
Total acidity minus total hydrochloric acid equals organic acids 
and acid salts. 
49 VII.-FECES 
Apparatus. 
Microscope 
Centrifuge 
Warm stage 
Ungraduated centrifuge tubes 
Test tubes, 6 inch 
Slides 
Covers 
f C.S.8E.C0 
^PATENTED 
Electrically Heated "Warm Stage" 
50 Reagents. 
Glacial acetic acid 
Ether 
Alcohol 
Gum guaiac 
Hydrogen peroxide 
Dilute hydrochloric acid 
Antiformin: 
Solution A. Sodium carbonate 15.0 grams 
Chlorinated lime 8.0 grams 
Water 100.0 c c. 
Solution B. Sodium hydroxide 15.0 grams 
Water 100.0 e c. 
Mix equal parts of A and B and filter. 
Blood-With a glass rod rub up a small portion of the 
stool in a test tube with water. Pour off the water solution into 
a clean test tube and boil for a few seconds. When cool, add 
one-third its volume of glacial acetic acid and shake well. Add a 
few cc. of ether and shake thoroughly. When the ether has 
separated, decant and add to the ether extract a few drops of 
guaiac tincture (prepared as directed for urine) and a few drops 
of hydrogen peroxide. A blue color results in the presence of 
blood. A reaction due to therapeutically administered iron and 
to blood from ingested meat must be excluded. 
Ova. Method of Yaoita-Take portions the size of a pea 
from several different parts of the feces. Place in a test tube and 
add 5 cc. of 25% antiformin. Mix thoroughly and warm, but do 
not boil. Allow to cool, add 5 cc. of ether and shake well. Filter 
through one layer of gauze and centrifuge the filtrate. Pour off 
the three layers which are formed from the fourth layer of sedi- 
ment. Treat the latter with dilute hydrochloric acid and a little 
ether. Shake well and centrifuge. Remove a small amount of 
the sediment with a capillary pipette; place on a slide and ex- 
amine at once, unstained, with the microscope. 
Parasites-Amebae are to be examined for as described in 
the section on bacteriology. The search for worms and worm 
segments is aided by washing the stool through a fine sieve. 
51